Tandem Affinity Purification (TAP) of Low-Abundance Protein Complexes in Filamentous Fungi Demonstrated Using Magnaporthe oryzae
Tandem Affinity Purification (TAP) of Low-Abundance Protein Complexes in Filamentous Fungi Demonstrated Using Magnaporthe oryzae
复制标题
使用 Magnaporthe oryzae 演示丝状真菌中低丰度蛋白质复合物的串联亲和纯化 (TAP)
DOI：
10.1007/978-1-0716-1613-0_8
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发表时间：
2021
影响因子：
--
通讯作者：
Wilson, RA.
中科院分区：
文献类型：
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作者：
Wilson, RA.
蛋白质-蛋白质相互作用是细胞结构和功能的基础。近年来，已经开发了许多用于鉴定参与控制各种生物途径的蛋白质复合物和组分蛋白质的方法。串联亲和纯化（TAP）结合质谱（MS）是一种强大的方法，其能够通过使用两种不同的表位标签进行两个连续的纯化步骤在温和条件下分离高纯度天然蛋白质复合物。在这个协议中，我们描述了一个TAP-MS方法，用于识别蛋白质-蛋白质相互作用，目前在真菌细胞中的非常低的水平。使用6xHis-3xFLAG双标签，我们使用高容量Ni 2+柱开始我们感兴趣的蛋白质的亲和纯化过程。与常规TAP方案中基于抗体的第一步纯化相比，这允许大大增加的样品输入，并提供大量高度浓缩和初步纯化的蛋白质复合物，以用于涉及FLAG免疫沉淀的第二纯化步骤。第二步极大地促进了在体内条件下捕获低水平相互作用的伴侣。我们的TAP-MS方法已被证明是安全的低丰度蛋白质复合物在生理条件下与高效率，特异性和经济的丝状真菌Magnaporthe oryzae的表征，并可能有利于基因功能和蛋白质组学研究在植物和其他研究领域。
Protein–protein interactions underlie cellular structure and function. In recent years, a number of methods have been developed for the identification of protein complexes and component proteins involved in the control of various biological pathways. Tandem affinity purification (TAP) coupled with mass spectrometry (MS) is a powerful method enabling the isolation of high-purity native protein complexes under mild conditions by performing two sequential purification steps using two different epitope tags. In this protocol, we describe a TAP-MS methodology for identifying protein-protein interactions present at very low levels in the fungal cell. Using the 6xHis-3xFLAG double tag, we start the affinity purification process for our protein of interest using high-capacity Ni2+columns. This allows for greatly increased sample input compared to antibody-based first-step purification in conventional TAP protocols and provides a large amount of highly concentrated and preliminarily purified protein complexes to be used in a second purification step involving FLAG immunoprecipitation. The second step greatly facilitates the capture of low-level interacting partners under in vivo conditions. Our TAP-MS method has been proven to secure the characterization of low-abundance protein complexes under physiological conditions with high efficiency, specificity, and economy in the filamentous fungusMagnaporthe oryzaeand might benefit gene function and proteomics studies in plants and other research fields.